Complementary somatic mutations of KCNJ5, ATP1A1, and ATP2B3 in sporadic aldosterone producing adrenal adenomas

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Complementary somatic mutations of KCNJ5,

ATP1A1, and ATP2B3 in sporadic aldosterone

producing adrenal adenomas

Ravi Kumar Dutta, Jenny Welander, Michael Brauckhoff, Martin Walz, Piero Alesina,

Thomas Arnesen, Peter Söderkvist and Oliver Gimm

Linköping University Post Print

N.B.: When citing this work, cite the original article.

Original Publication:

Ravi Kumar Dutta, Jenny Welander, Michael Brauckhoff, Martin Walz, Piero Alesina, Thomas

Arnesen, Peter Söderkvist and Oliver Gimm, Complementary somatic mutations of KCNJ5,

ATP1A1, and ATP2B3 in sporadic aldosterone producing adrenal adenomas, 2014,

Endocrine-Related Cancer, (21), 1, L1-L4.

http://dx.doi.org/10.1530/ERC-13-0466

Copyright: BioScientifica

http://www.bioscientifica.com/

Postprint available at: Linköping University Electronic Press

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-106692

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Complementary somatic mutations of KCNJ5, ATP1A1 and

ATP2B3 in sporadic aldosterone producing adrenal

adenomas

Ravi Kumar Dutta1_{, Jenny Welander}1_{, Michael Brauckhoff}2,3_{, Martin Walz}4_{, Piero Alesina}4_{, Thomas} Arnesen2,5_{, Peter Söderkvist}6,#_{, Oliver Gimm}7,#,*_{; #These authors contributed equally}

1_{Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University,}

Linköping, Sweden

2_{Department of Surgery, Haukeland University Hospital, Bergen, Norway}

3_{Department of Surgical Sciences, Institute of Medicine, University of Bergen, Bergen, Norway}

4_{Klinik für Chirurgie and Zentrum für Minimal Invasive Chirurgie, Klinikum Essen-Mitte, Essen, Germany} 5_{Department of Molecular Biology, Institute of Medicine, University of Bergen, Bergen, Norway}

Department of Clinical Genetics, County Council of Östergötland, Linköping, Sweden

Department of Surgery, County Council of Östergötland, Linköping, Sweden

*Corresponding author

Oliver Gimm, Head, Division of Clinical Sciences, Department of Clinical and Experimental Medicine Linköping University, SE-58183 Linköping, Sweden

Telephone: +46 10-103 80 41, Fax: +46-10-103 35 70, E-mail address: oliver.gimm@liu.se

Short title: Complementary mutations in Conn tumors

Keywords: Mutations, somatic, KCNJ5, ATP1A1, ATP2B3, aldosterone, adrenal adenoma Word count: 1013

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Dear Editor,

Primary aldosteronism (PA) is the most common form of secondary hypertension, accounting for 8-13% among hypertension patients (Mulatero, et al. 2013). It is characterized by constitutive production of aldosterone by the adrenal cortex. Among the subtypes of PA, aldosterone producing adenomas (APAs), also known as Conn tumors, are characterized by tumors in the adrenal cortex and account for 30-40% of the cases. The two most important physiological stimuli of aldosterone secretion are angiotensin II and serum potassium. Decrease in blood volume activates the renin-angiotensin system in which angiotensin II signals via the angiotensin receptor. The K+_{concentration across the membrane sets the resting}

membrane potential. Hyperkalemia causes depolarization of the membrane and generates an action potential to open a voltage gated Ca2+_{channel. In both cases, enhanced intracellular Ca}2+_{provides the}

normal signal for aldosterone production. In APAs, autonomous production of aldosterone is found independently of angiotensin II.

Recently, next generation sequencing has revealed novel genes frequently mutated in APAs: KCNJ5, ATP1A1 and ATP2B3 (Beuschlein, et al. 2013; Choi, et al. 2011; Mulatero et al. 2013; Taguchi, et al. 2012). In these pivotal studies, mutations in KCNJ5, encoding an inwardly rectifying K+_{channel, were}

identified in about 30-45% of patients. The K+_{channel encoded by KCNJ5 exists both as homo-tetramer}

and as a hetero-tetramer with another potassium channel encoded by KCNJ3. The latter has been found more active than homo-tetramers (Choi et al. 2011). More recently, mutations in ATP1A1 (encode a Na+/K+ pump ATPase α subunit) and ATP2B3 (plasma membrane Ca2+_{ATPase) were reported, each of}

which appears in about 6% and 2% of the tumors, respectively (Beuschlein et al. 2013). In the present study, we investigated KCNJ5, KCNJ3, ATP1A1 and ATP2B3 for mutations in a series of 35 consecutive patients with sporadic APAs from Norway, Sweden and Germany (protocols and primers available on request).

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We found frequent somatic mutations in KCNJ5, ATP1A1 and ATP2B3. No mutations were identified in KCNJ3 which is in agreement with previous reports (Beuschlein et al. 2013; Choi et al. 2011; Taguchi et al. 2012).

Regarding KCNJ5 (NM_000890.3), 11 (31%) missense mutations were identified. Seven mutations were at c.451G>A (p.Gly151Arg), one at c.451G>C (p.Gly151Arg) and three at c.503T>G (p.Leu168Arg) (Fig. 1a, 1b & 1c, respectively). The overall mutation frequency was in agreement with previous reports (Choi et al. 2011; Taguchi et al. 2012). Notably, the somatic mutations G151R and L168R are situated on the highly conserved Glycine-Tyrosine-Glycine (GYG) motif of the selective filter and the second transmembrane (TM) domain of KCNJ5, respectively (Heginbotham, et al. 1992). The GYG motif in the extracellular loop of all four subunits of the KCNJ5 channel forms the narrowest part of the pore. Both mutations abolish the highly conserved region of the GYG motif. In in vitro studies, it appears that all mutations potentially lead to a loss of ion selectivity of the channel protein (Choi et al. 2011).

Furthermore, reduction of inward K+_{current results in enhanced depolarization of the adrenal cells which}

leads to activation of voltage gated Ca2+_{channel. An increase in intracellular Ca}2+_{is associated with}

higher aldosterone production.

Regarding ATP1A1, two missense variants (6%) were identified at c.311T>G (p.Leu104Arg) (Fig. 1d). Concerning ATP2B3, three inframe deletions (9%) were found, two of c.1272_1277delGCTGGT (p.Leu425-Val426del ) and one of c.1281_1286delGGCTGT (p.Arg428-Val429del) (Fig. 1e & 1f). The overall mutation frequencies were slightly higher than in one previous report (Beuschlein et al. 2013) which may be due to small sample size. Of note,we identified the novel mutation

c.1281_1286delGGCTGT in ATP2B3.

The protein encoded by both genes ATP1A1 and ATP2B3 exchanges K+_{and Ca}2+_{ions, respetively, by}

hydrolysis of one ATP (Di Leva, et al. 2008; Kaplan 2002). On the crystal structure of ATP1A1, the mutant L104R is located in the transmembrane α helix M1, which has been suggested to interact and cooperate in K+_{ion binding and gating by interaction with Glu334 (Morth, et al. 2007). It has been found}

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(Hajnoczky, et al. 1992). Since Ca2+_{ion pumps are highly conserved, we used sarcoplasmic reticulum}

type Ca2+ _{ATPase (SERCA) to project the mutations. The deletions 425Ala_426Val and 428Ala_429Val}

corresponds to 303Ala_304Val and 306Ala_307Ile (Fig. 1g). The PEGLP motif after Ile307 is a key motif for ion gating and is highly conserved among the P type pumps (Di Leva et al. 2008). Mutations potentially lead to the distortion of this Ca2+_{binding region. Notably, in both ATPase genes, the mutation}

abolishes Glu334 and Glu309 in ATP1A1 and ATP2B3 that are crucially important for ion gating.

Functional ex vivo studies of the role of the loss of function mutations in the ATPase genes (Beuschlein et al. 2013) showed substantially higher levels of depolarization in the mutated samples.

In this study, the expression of KCNJ5 at the mRNA level was found to be significantly lower in mutated samples (P=0.02) (Fig. 1h). This finding is in disagreement with previous results (Boulkroun, et al. 2013; Taguchi et al. 2012). The reason for this discrepancy might be the rather small sample size. In contrast to KCNJ5, the mRNA expression levels of ATP1A1 and ATP2B3 were not affected by mutational status (Fig. 1i & 1j, respectively). This is in agreement with previous results (Beuschlein et al. 2013).

Clinical characteristics of the patients are shown in Table 1. In contrast to patients with KCNJ5 mutations, ATPase mutated APAs were predominantly found in males (Table 1). There was no statistically

significant difference concerning the age of patients having APAs with different mutations (Fig. 1k). While the tumor size of APAs with somatic KCNJ5 mutations was almost twice the size of APAs with either somatic ATP1A1 and ATP2B3 mutations, this difference was not statistically significant (Fig. 1l). No conclusions could be drawn from the preoperative aldosterone levels (Fig. 1m).

In conclusion, somatic mutations found in KCNJ5, ATP1A1 and ATP2B3 appear to be driving forces for a higher aldosterone production and proliferations of glomerulosa cells. All mutations found in this study were complementary to each other (Fig. 1n) indicating that multiple genes may contribute independently to the formation of APAs.

Ravi Kumar Dutta1_, Jenny Welander1_,

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Michael Brauckhoff2,3_, Martin Walz4_, Piero Alesina4_, Thomas Arnesen2,5_, Peter Söderkvist6,#_, Oliver Gimm7,#,*

Linköping, Sweden

2_{Department of Surgery, Haukeland University Hospital, Bergen, Norway}

3_{Department of Surgical Sciences, Institute of Medicine, University of Bergen, Bergen, Norway}

4_{Klinik für Chirurgie and Zentrum für Minimal Invasive Chirurgie, Klinikum Essen-Mitte, Essen, Germany} 5_{Department of Molecular Biology, Institute of Medicine, University of Bergen, Bergen, Norway}

Department of Clinical Genetics, County Council of Östergötland, Linköping, Sweden

Department of Surgery, County Council of Östergötland, Linköping, Sweden

#These authors contributed equally

*Corresponding author

Oliver Gimm, Head, Division of Clinical Sciences, Department of Clinical and Experimental Medicine Linköping University, SE-58183 Linköping, Sweden

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Declaration of interest

The authors declare that they have no conflict of interest.

Acknowledgements

We thank Annette Molbaek and Åsa Schippert for technical assistant. This work was supported by grants from Linköping University to both Oliver Gimm and Peter Söderkvist.

References

Beuschlein F, Boulkroun S, Osswald A, Wieland T, Nielsen HN, Lichtenauer UD, Penton D, Schack VR, Amar L, Fischer E, et al. 2013 Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension. Nat Genet 45 440-444, 444e441-442.

Boulkroun S, Golib Dzib JF, Samson-Couterie B, Rosa FL, Rickard AJ, Meatchi T, Amar L, Benecke A & Zennaro MC 2013 KCNJ5 mutations in aldosterone producing adenoma and relationship with adrenal cortex remodeling. Mol Cell Endocrinol 371 221-227.

Choi M, Scholl UI, Yue P, Bjorklund P, Zhao B, Nelson-Williams C, Ji W, Cho Y, Patel A, Men CJ, et al. 2011 K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension. Science 331 768-772.

Di Leva F, Domi T, Fedrizzi L, Lim D & Carafoli E 2008 The plasma membrane Ca2+ ATPase of animal cells: structure, function and regulation. Arch Biochem Biophys 476 65-74.

Hajnoczky G, Csordas G, Hunyady L, Kalapos MP, Balla T, Enyedi P & Spat A 1992 Angiotensin-II inhibits Na+/K+ pump in rat adrenal glomerulosa cells: possible contribution to stimulation of aldosterone production. Endocrinology 130 1637-1644.

Heginbotham L, Abramson T & MacKinnon R 1992 A functional connection between the pores of distantly related ion channels as revealed by mutant K+ channels. Science 258 1152-1155. Kaplan JH 2002 Biochemistry of Na,K-ATPase. Annu Rev Biochem 71 511-535.

Morth JP, Pedersen BP, Toustrup-Jensen MS, Sorensen TL, Petersen J, Andersen JP, Vilsen B & Nissen P 2007 Crystal structure of the sodium-potassium pump. Nature 450 1043-1049.

Mulatero P, Monticone S, Rainey WE, Veglio F & Williams TA 2013 Role of KCNJ5 in familial and sporadic primary aldosteronism. Nat Rev Endocrinol 9 104-112.

Taguchi R, Yamada M, Nakajima Y, Satoh T, Hashimoto K, Shibusawa N, Ozawa A, Okada S, Rokutanda N, Takata D, et al. 2012 Expression and mutations of KCNJ5 mRNA in Japanese patients with aldosterone-producing adenomas. J Clin Endocrinol Metab 97 1311-1319.

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Table 1: Clinical characteristic of 16 APA patients with different mutations in KCNJ5, ATP1A1 and ATP2B3

sample age (years) sex preop aldo (ng/l) size (mm) gene cDNA bp

L1 39.1 M 580 7 KCNJ5 c.451G>A L15 49.1 F 290 10 ATP2B3 c.1281_1286delGGCTGT L37 58.0 M 470 10 ATP1A1 c.311T>G L58 45.9 F 530 10 KCNJ5 c.451G>C L70 32.1 F 980 25 KCNJ5 c.503T>G B1 64.3 M 1246 11 ATP2B3 c.1272_1277delGCTGGT B2 37.7 F 1675 17 KCNJ5 c.503T>G B9 36.4 F 1013 37 KCNJ5 c.451G>A B17 47.7 M 1078 17 KCNJ5 c.451G>A G1 54.9 M 300 26 KCNJ5 c.451G>A G2 60.5 M 350 15 ATP1A1 c.311T>G G3 69.4 F 329 19 KCNJ5 c.503T>G G4 60.9 M 184 15 KCNJ5 c.451G>A G6 59.1 F NA 11 KCNJ5 c.451G>A L131 44.2 F 390 15 KCNJ5 c.451G>A L141 44.8 M 460 12 ATP2B3 c.1272_1277delGCTGGT M=Male F=Female NA=Not available mm=millimeters ng= Nanogram l= liter

Sequences of blood DNA showing no mutation (WT) and mutated tumor DNA showing the following somatic missense mutations c.451G>A (a), c.451G>C (b) and c.503T>G (c). Normal blood and mutated tumor DNA sequences regarding ATP1A1 (c.311T>G) (d), c.1272_1277delGCTGGT ATP2B3 (e) and c.1281_1286delGGCTGT (f), respectively).

Alignment of plasma membrane Ca2+_{ATPase pumps and sarcoplasmic reticulum type Ca2}+_{ATPases (g).}

Colored region are conserved among them. The arrow indicates the deleted residues in our cases. The PEGLP motif is conserved among all p-type pump. It is a key factor for ion gating.

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mRNA expression of KCNJ5 in APAs with mutation (Mut KCNJ5) and without KCNJ5 mutation (KCN5-) (h(KCN5-). The mRNA levels of mutated KCNJ5 were significantly lower (p=0.02(KCN5-). Expression of ATP1A1 mRNA of APAs with (Mut ATP1A1) and without mutation (ATP1A1-) (i). Expression of ATP2B3 mRNA in APAs with (Mut ATP2B3) and without mutation (ATP2B3-) (j).

Age of patients with APAs with regard to the somatic mutation (KCNJ5, ATP1A1 and ATP2B3)(k). Diameter of APAs with regard to the somatic mutation (l). Comparison of aldosterone levels of the patients with APAs with regard to the somatic mutation (KCNJ5, ATP2B3 and ATP1A1) (m). Lines show the mean value of each group.

Complementary mutations of KCNJ5, ATP2B3 and ATP1A. Mutation frequencies of 31% for KCNJ5, 9% for ATP2B3 and 6% for ATP1A1 were observed in our cohort (n).